Integrins are ?/ heterodimeric cell surface receptors that, via their ability to exist in multiple conformations, regulate a diverse array of biological processes ranging from organ development to immune regulation to hemostasis. Proteins binding to either the extracellular or cytoplasmic face of an integrin can effect long-range conformational changes that are transmitted across the plasma membrane. Bi-directional signal transmission from outside to inside initiates cell signaling cascades supporting cell migration and survival, while binding of specific proteins to the cytoplasmic tails of the integrin (inside-ot signaling) induces long-range conformational rearrangements within the ectodomains that convert it from a resting to an activated (ligand-binding) competent state. Despite many studies over the past 25 years, exactly how inside-out and outside-in signaling is controlled through the transmembrane and cytoplasmic domains are still not understood at the structural level. Development of novel therapeutics free of confounding side effects requires an atomic level molecular understanding of these processes. We recently found that deleting the C-terminus of the ?-subunits in integrins, including the platelet ?IIb3 and leukocyte aL2, has a dramatic ad unexpected effect on the ability of integrins to undergo conformational change in response to inside-out signals. Aim 1 of this proposal will focus on defining this novel role of integrin a-subunit cytoplasmic domains, especially their less-conserved membrane distal region, in regulating integrin inside-out activation. Multiple independent approaches, including comprehensive mutagenesis, cysteine scanning accessibility, disulfide crosslinking, and cysteine fluorescence labeling of full-length integrins, will be employed to examine restructuring of integrin transmembrane and cytoplasmic domains in response to inside-out and outside-in signaling in coordination with the multiple active conformations of the ectodomain. Aim 2 will focus on a second important issue: namely, the observation that most RGD (Arg-Gly-Asp)-mimetic integrin antagonists in clinical trials or in clinical use for treating thrombosis or cance have the undesirable side effect of inducing thrombocytopenia and in some cases, thrombosis or enhanced tumor growth and angiogenesis. These effects appear to be due to drug-induced conformational changes leading to integrin activation and creation of neo-epitopes capable of inducing an immune response. Building on our recent structural studies, we will define at the atomic level the critical structural changes that take place when an integrin binds either small or large ligands. Findings made will be used to guide the development of novel integrin antagonists that block function without altering integrin conformation. Together, these complementary Specific Aims will reveal the molecular basis for ligand-induced integrin conformational change and provide the structure-based understanding of bidirectional integrin signaling necessary to facilitate development of novel integrin antagonists with potentially improved therapeutic efficacy.